E . KATCHALSKI, R. H. 5058 H. EDELHOCH,
[CONTRIBUTION FROM
THE
MAYBURY,
W. L. HUGHES, JR.,
AND
J. T. EDSALL Vol. 7 5
UNIVERSITYLABORATORY OF PHYSICAL CHEMISTRY RELATED TO MEDICINEAND PUBLIC HEALTH, HARVARD UNIVERSITY ]
Dimerization of Serum Mercaptalblrmia in Presence of Mercurials. I. Kinetic and Equilibrium Studies with Mercuric Saltslapb~c BY HAROLD EDELHOCH, EPHRAIM QTCHALSKI, JOHN
ROBERTH. MAYBURY, WALTER L. HUGHES, JR., T. EDSALL’~
AND
RECEIVED APRIL 13, 1953 Human serum mercaptalbumin (ASH) contains one sulfhydryl group per molecule. In the presence of a mercuric salt H g X p w h e r e X denotes chloride or acetate-mercury is very rapidly bound to the sulfhydryl group, forming the compound ASHgX. This reacts much more slowly with another molecule of mercaptalbufnin to give the mercury dimer, ASHgSA, ke the reaction probably being ASH ASHgX f;r ASHgSA H + X-. The velocity constants ka and k--2 and the k-r “apparent equilibrium constant” K’ = (ASHgSA)/(ASH)(ASHgX) have been studied by light scattering measurements as functions of pH, temperature and the concentrations of various added anions. The calculation of weight average molecular weights from light scattering in systems containing mercaptalbumin monomer and dimer is discussed, with particular reference t o the effects of interactions due to net charge on the protein Repeated experiments have demonstrated the reversibility of the reaction. Titration of ASH with increasing amounts of HgXt shows that dimer formation a t equilibrium is a maximum when the molar ratio (Hg)/(ASH) = 0.5; the curve is symmetrical around this point, no dimer being present if (Hg)/ (ASH) is zero or unity. The equilibrium constant K’ is near 3 X lo4 1. mole-’ a t 25’, and is nearly independent of pH from 4.7 t o 6; it increases with rise of temperature. The kinetics of dimer formation can be described on the assumption that the reaction is approximately second order in the protein concentration, when equal amounts of ASH and ASHgX are present. The velocity constant of dimerization, ks, is near 40 1. mole-’ m h - 1 at 25’, for the isoelectric protein a t pH 4.75; it decreases to 0.7 a t p H 6, probably owing to the electrostatic repulsion of the negatively charged protein molecules a t this pH. The energy of activation for dimerization is 18-20 kcal./mole, and the calculated standard entropy of activation is very small, in contrast to the large negative values generally found for reactions in which two molecules are joined. Added halide ions, except fluoride, decrease K’, the relative amounts of C1-, Br- and I- required to produce a given decrease being approximately in the ratio 2000:80:> kz. Thus, in the studies to be discussed mole Hg/mole albumin. As soon as a reading could here, we are concerned with the velocity constants be taken on this solution-about one minute after kz and k-2. the mercury was added-the value of I280 was found It could also be shown that, in a solution of to have decreased to the value characteristic of the fixed total protein concentration, pH, mercury and monomer solution (see reaction I11 above) and it resalt concentrations, the same final equilibrium mained steadily a t this value for the next two weeks, state was attained when the reaction was allowed (19) The use of fluoride senred two purposes: (1) since mercuric to proceed in either direction. An example is given Euoride is an ionic compound, unlike the other mercury halider, the in Fig. 3. Mercuric chloride was added to a con- presence of fluoride ion serves to maintain the ionic strength while at with the albumin for mercury, which centrated mercaptalbumin solution, the final con- the same time it does not compete (2) the presence of fluoride inhibite would inhibit dimer formation: centration of protein being 0.162 g./ml. and the bacterial growth, of which there is a considerabe risk in such prolonged ratio HgClz/albumin being 0.45. The concentrated experiments on protein solutionr at room temperature.
T,OM
H.
RDELHOCII,
E. KATCHALSKI, r
